The present invention relates to probes comprising fluorescent compounds. The present compositions may be employed in the detection and quantitation of analytes such as polypeptides, polynucleotides, hormones and drugs. In particular, the invention pertains to fluorescent compounds and their use as detection groups in electrophoretic probes.
The need to determine many analytes (for example multiple pathogens or multiple genes or multiple genetic variants) in blood or other biological fluids has become increasingly apparent in many branches of medicine. Most multi-analyte assays, such as multiplexed assays that detect, e.g., multiple nucleic acid sequences, involve multiple steps, have poor sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold differences and some require sophisticated instrumentation.
Fluorescent compounds are widely used as labels in a variety of diagnostic procedures. Fluorescent compounds are linked to other entities to form probes that are used to assay for a wide variety of materials of interest. In some applications it is important that fluorescent compounds employed as labels have different spectral properties. Such properties include wavelength of excitation, wavelength of emission, quantum yields, Stokes shift, and so forth.
U.S. Pat. No. 6,008,379 discloses aromatic substituted xanthene dyes.
U.S. Pat. No. 6,080,852 discusses 4,7-dichlororhodamine dyes.
Unsymmetrical fluorescein derivatives are disclosed in U.S. Pat. No. 4,439,356.
Xanthene dyes having a fused (C) benzo ring are discussed in U.S. Pat. No. 4,945,171.
4,7-Dichlorofluorescein dyes as molecular probes are discussed in WO 94/05688.
U.S. Pat. No. 4,351,760 discloses alkyl substituted fluorescent compounds and polyamino acid conjugates.
U.S. Pat. No. 4,318,846 discusses ether substituted fluorescein polyamino acid compounds as fluorescers and quenchers. PCT WO 97/39064 discloses fluorinated xanthene derivatives.
U.S. Pat. No. 5,807,682 describes probe compositions for detecting a plurality of nucleic acid targets.
In one embodiment the present invention is directed to electrophoretic tag probes, and sets of such probes, wherein a probe comprises a releasable portion that is separately detectable on the basis of mobility and a detectable moiety that is a compound of the formula: 
wherein:
Z is H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aromatic, substituted aromatic, phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, heterocyclic, substituted heterocyclic, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, imino, phosphorus containing species such as, e.g., phosphate, phosphite, and the like, polymer chains of from about 2 to about 500, preferably, about 5 to about 100 monomer units such as, e.g., nucleotides, amino acids, amides, esters, ethers, amines, and so forth,
A is O, N+(R1)(R2) wherein R1 and R2 are independently H, lower alkyl, substituted lower alkyl, and the like,
D is OH, OR3 wherein R3 is lower alkyl, substituted lower alkyl, aryl, substituted aryl, and the like, N(R1)(R2) wherein R1 and R2 are independently H, lower alkyl, substituted lower alkyl,
W1, W2, W3, W4, W5 and W6 are independently H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aromatic, substituted aromatic, phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, heterocyclic, substituted heterocyclic, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, imino, and so forth,
X1-X4 are independently H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aromatic, substituted aromatic, phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, heterocyclic, substituted heterocyclic, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, imino, and so forth,
wherein W2 and W3 may be taken together to form one or more rings comprising 4 to 14 atoms, preferably, 4 to 8 atoms, more preferably, 5 to 7 atoms, usually carbon atoms, and comprising 1 to 7 unsaturations, usually, 1 to 4 unsaturations, such as, e.g., benzo (from benzene), naptho (from naphthalene), anthro (from anthracene), and the like, and
wherein W4 and W5 may be taken together to form a ring comprising 4 to 14 atoms, preferably, 4 to 8 atoms, more preferably, 5 to 7 atoms, usually carbon atoms, and comprising 1 to 7 unsaturations, usually, 1 to 4 unsaturations, such as, e.g., benzo (from benzene), naptho (from naphthalene), anthro (from anthracene), and the like,
The electrophoretic probes may be used in methods for simultaneously determining multiple analytes in a sample suspected of containing the analytes. In the method a combination is provided comprising the sample and an electrophoretic tag probe comprising a releasable portion that is separately detectable on the basis of mobility and the presence therein of a fluorescent compound in accordance with the invention. The electrophoretic tag probe, or e-tag probe, is involved in a binding event that is related to the presence of the analyte in the sample. The combination is treated with reagents under conditions sufficient to release the releasable portion, forming an e-tag reporter. The presence and/or amount of the released e-tag reporter is detected and is related to the presence and/or amount of the analyte in the sample.